Biosensors

Wei-Chih Wang
Southern Taiwan University of
Technology

w. wang 1
 Biosensors

Analyte: substances to be measured

Small molecules: sugars, cholestrol, glutamic acid, phosphate, etc.
Macro molecules: amino acid (DNA, RNA), peptide (protein, antibody, enzyme)
Receptor: sensing element responds to substance being measured, interaction is
highly selective.
Enzyme, antibody, cells
Transducer: device converts physical or chemical changes due to analyte-receptor
reactions to eletronic signals whose magnitude is proportional to the amount of the
analyte
Electrochemical (potentiometric, voltammetric, conductimetric)
Optical (fluorescence, absorbance, light scattering, refractive index)
Field effect transistor
Mechanical, thermal, Piezoelectric,
w. SAW,
wang Magnetic 2
Signal processor: amplifier, filtration, correlation etc.
 Transduction Methods: Electrochemical

-Potentiometry : Electrode (reference)

the measurement of a cell
potential at zero currents Glass membrane (ion-sensitive)
Electrode

-Amperometry Working Electrode

in which an oxidizing (or reducing)
potential is applied between the cell
electrodes, and the cell current is
measured Reference electrode Auxiliary electrode

sensor
-Conductometry Vref
Vout
where the conductance of the cell
is measured
Bridge circuit

w. wang 3
5 cm 5 mm 10mm
 Ion Selective Field Effect Transistor

ISFETs sense the concentration (activity level) of a particular ion in a solution.

w. wang 4
Gate metal is replaced with an ion selective membrane.
Current from source to drain related the gate potential
When there is a high concentration of positive ions in the solution, a lot of them will
accumulate on the gate, widening the channel between the source and drain. With
a low concentration of positive charged ions, the channel will be narrow.
w. wang 5
 (Fronherz, 2001)

The chip with cultured HEK293 cells is illuminated with blue

light and observed through a filter that transmits some light
reflected from the chip and the emission of green
fluorescent protein used as a reporter for the transfection
with maxi-KCa channels. w. wang 6
Enzyme-based biosensor
glucose oxidized based sensor

w. wang 7
 Optical Sensors for Chemical or
Biochemical Sensing
- Light application for monitoring tissues and cells:

- A multi-parameter sensor-chip-based system

Advantages:
- sensitivity
- simple and compact
- noninvasive !
w. wang 8
 Integration of optical sensors into
microfluidic system

- For use in optical microscopy and cellular biology

- Measure the chemical properties of cells or monitor
chemical changes that take place within cells as they respond
to external changes in their environment and to internal
changes that occur in embryonic cells as an organism develops
(on-line monitoring)

w. wang 9
 Transduction Methods : Optical

Types of measurements Changes measured

-Absorbance(
Oligo260nm, Peptide280nm) -Intensity
-Fluorescence -Frequency
-Refractive index -Phase shift
-Light scattering -Polarization

Types of components
-Fiber optics
-Wave guides
-Photodiode
-Spectroscopy
-Charge coupled device (CCD)
-Single photon APD
-Interferometers

w. wang 10
 Optical Biosensor Basing on Evanescent Wave

Evanescent Wave Separation between surface

and bulk

Noise reduction due to small

observation volume

Detector
Light Source
Surface Plasmon
Resonance (SPR) Prism

Evansescent wave
High index (glass)
Low index (Au or Ag) D1 D2
Sensing layer
Flow cell

Biotinylated capture probe

Evanescent Wave Fiber
Optic Sensor
Streptavidin
Biotin
Biotinylated Fiber
w. wang 11
Laser Induce Fluorescence (LIF) Based Detection

Detector

Pinhole

Extremely High Sensitivity

Band pass filter Very small probe volume (< 10-15L)

Dichoric
-Less Raman scattering noise from solution
Laser -Less background luminescence
beamsplitter
Monochromatic excitation
Objective -Noise from light source can be efficiently
filtrated

Enhance Signal to Noise Ratio

w. wang 12
 Regenerable biosensor

(Sapsford K 2002)

Electrochemical control of the binding and dissociation

of antibody-antigen
Total internal reflectance fluorescence (TIRF)
spectroscopy sensing techniques
w. wang used 13
 OPTICAL WAVEGUIDE
LIGHTMODE SPECTROSCOPY

Microvaccum Inc.

w. wang 14
 Theory and Models

Electromagnetic field
distribution for a zeroth
Ray-optic representation mode
of a coupled and guided Microvaccum Inc.

wave

w. wang 15
 Microvaccum Inc.

w. wang 16
 Microvaccum Inc.

w. wang 17
 Immunosensor

Microvaccum Inc.

Monomolecular chemoresponsive coating, which consists of

immobilized antibody (Ab) molecules, that bind the
corresponding antigen (Ag) molecules.
w. wang 18
 Chemosensor

Microvaccum Inc.

With a typically 0,1-1 mm thick chemoresponsive layer

whose refractive index is changed by binding the analyte
molecules.
w. wang 19
 Proposed Optical Biosensor
Components
Waveguide
Microfluidic channels
How it works
based on total internal
reflection

w. wang 20
 What to detect?
DNA
Antibodies
Other molecules or chemicals

w. wang 21
 Biochemical Sensors
- Oxygen sensor- oxygen regent sensor, (immobilized hemoglobin which
changes color with absorbed oxygen), oxygen reflectance spectroscopy .
- Carbon dioxide sensor- measured by monitoring pH of a
bicarbonate-carbonic acid equilibrium mixture, according to
the mass-reaction equation.
- Surface reaction measurement (i.e. chemicaly linked to waveguide in a
manner which preserves the specific binding activity, transmission
properties of guide are used to meaure antibody-antigen reaction) light
scattering, fluorescence, absorption
- Flow sensor (laser-Doppler, interferometer)
- Glucose detector (viscosity, absorption, reflection)
- Concentration
- pH sensor (hydrogen ion-pearmable membrane enclosing a colorimetric
pH indicator- optical sensor monitoring changes in reflectance as dye
responds to pH changes)
w. wang 22
- Acidity sensor
- Turbidity (or scattering) measurement (determination of
impurities in liquids and the measurement of particle size- oil
in water monitoring reflectance probe)

w. wang 23
Applications for Medical Instrumentation
- Endoscopes
- Laser surgery and therapy
- Angioplasty
-Temperature sensors (hyperthermia, cardiac monitoring, by
thermodilution, tissue thermal damage monitoring)
-Pressure sensor (cardiovascular, intracranial,neurology
urodynamics)
- Chemical sensors:blood oximetry, pH, pO2, pCO2
(metabolic and respiratory problems, blood an tissue oxygen
content, oxygen,hemoglobin dissociation curve)

w. wang 24
 Material Requirements
(Optical)
Low loss
absorption
scattering
Low fluorescence
High refractive index
Easy to fabricate
w. wang 25
 Material Requirements
(Fluidic)
Biocompatible

Hydrophobic/Hydrophilic

Sufficient bond strength with optic components

Low temperature bonding necessary

w. wang 26
 Biocompatibility
- The interface between live cells and sensor is one of the
key issues of the design
- Study of biocompatible materials (i.e. PEG, PEO avoid adsorption
of proteins, clogging with cells, immune response etc.)
- Study of gas or molecule absorbing materials (i.e. thermally
responsive biopolymer ppNIPAM, enzyme,antibody, cells)

w. wang 27
 Possible Materials
Present
Si/Si3N4
glass/ion-exchange
PDMS
Future
PMMA
Mylar
Teflon w. wang 28
Material Properties-Ion Exchange
Glass
Loss: 0.0006/cm
Fluorescence: 510112
(arb)
n = 1.47
Biocompatible: Yes
Hydrophilic

w. wang 29
 Material Properties - PDMS
Loss: 0.0008/cm
Fluorescence: 36383
(arb)
n = 1.5
Biocompatible: Yes
Hydrophobic

w. wang 30
 Fabrication Techniques
Bulk micro-machining
- Wet etching
- Vapor-phase etching
- Plasma/reactive ion etching
- molding
- Laser etching
- screen printing
Surface micro-machining
- Thin film process (sol-gel, wet etching, dry etching, film
deposition, lift-off patterning, epitaxial growth etc.)
- sacrificial process (phase-change release, plasma etching,
sacrificial layer)
- bonding process (anodic bonding, silicon fusion etc.)
- laser etching
- E-beam
- LIGA w. wang 31